Method of preparing a cryoprecipitated suspension and use thereof

ABSTRACT

This invention concerns a method of preparing a cryoprecipitated suspension containing fibrinogen and Factor XIII useful as a precursor in the preparation of a fibrin glue which involves (a) freezing fresh frozen plasma from a single donor such as a human or other animal, e.g., a cow, sheep or pig, which has been screened for blood transmitted diseases, e.g., one or more of syphilis, hepatitis or acquired immune deficiency syndrome at about -80 DEG  C. for at least 6 hours, preferably for at least about 12 hours; (b) raising the temperature, so as to form a supernatant and a cryoprecipitated suspension containing fibrinogen and Factor XIII; and (c) recovering the cryopricipitated suspension.

BACKGROUND OF THE INVENTION

This application is a continuation of U.S. Ser. No. 893,338, filed Aug.5, 1986, now abandoned, which is a divisional of U.S. Ser. No. 688,601,filed Jan. 3, 1985, now U.S. Pat. No. 4,627,879, issued Dec. 9, 1986which is a continuation-in-part of U.S. Ser. No. 648,752, filed Sept. 7,1984, now abandoned, the contents of which are hereby incorporated byreference into the present application.

Within this application several publications are referenced by arabicnumerals within parentheses. Full citations for these references may befound at the end of the specification immediately preceding the claims.The disclosures of these publications in their entireties are herebyincorporated by reference into this application.

A major technical advance in surgery underutilized in this country hasbeen the clinical application of fibrin glue. Clinical reports(1,3,8,9,10,12,14,16,17) document the utility of this concentratedadhesive, which duplicates the biological process of the final stage ofnormal coagulation (FIG. 1). Fibrin glue has been used to controlbleeding from liver lacerations and traumatized spleens (3,14). Otheruses include sealing sewn or stapled tracheal and esophageal anastomosesas well as persistent air leaks or lacerations of the lung (1,12).Bronchial fistulas have been successfully closed using fibrin adhesive.A useful application has been in vascular surgery. An important featureis its ability to achieve hemostasis at vascular anastomosesparticularly in areas which are difficult to approach with sutures orwhere suture placement presents excessive risk (1,10,12,16). Bleedingfrom needle holes or small arterial tears which cannot be controlled bysuturing alone usually can be sealed by judicious fibrin glueapplication (1). It has been especially helpful in obtaining hemostasisin heparinized patients or those with coagulopathy (1,17). Furthermore,fibrin glue impregnation permits the use of porous knitted grafts, evenin anticoagulated patients, eliminating bleeding which has prevented thewidespread use of these porous grafts in open heart surgery (1,8,9,16).

Various techniques have been described to pretreat porous vascularprostheses. Many are complicated, time consuming, expensive proceduresand often render prostheses stiff and non-yielding (7,18). A previouslydescribed highly effective method using a cryoprecipitate preparation(6) was criticized because of its high cost (11).

Haverich et al. (8) reported that fibrin presealing allows the use ofhigh porosity knitted Dacron prostheses even in heparinized patients. Ahighly porous fabric, with its superior healing characteristics, offersthe potential for a lower incidence of right ventricular conduitobstruction (8). Fibrin presealed grafts are no more thrombogenic, andmay be less so, than untreated grafts or those pretreated with blood(9,18). Highly porous fabrics have superior handling characteristicscompared to low porosity grafts, and the use of fibrin adhesive couldmake low porosity woven Dacron grafts obsolete.

Despite generalized acceptance and use in Europe as a tissue sealant andhemostatic agent, fibrin glue has received little attention in theUnited States. In large part, this stems from the 1978 U.S. Food andDrug Administration ban (13) on the sale of commercially preparedfibrinogen concentrate made from pooled donors, e.g., as in Schwarz, etal., U.S. Pat. Nos. 4,298,598 (1981), 4,362,567 (1982) or 4,414,976(1983), because of the risk of transmission of viral infection, inparticular hepatitis B (2). In addition, the recent appearance ofAcquired Immune Deficiency Syndrome also a major health concern, makesit unlikely that there will be a change in this policy in theforeseeable future (4).

Concentrated fibrinogen can be prepared with minimal risk of diseasetransmission from a patient's own blood (5,19). Although this techniqueobviously eliminates the risk of blood transmitted viral infection, itrequires anticipating surgery at least two days in advance so that theautologous blood can be drawn and prepared in time. In addition, itrequires the patient to donate at least one unit of blood and may resultin the need for blood transfusion to replace donated blood. Furthermore,it is not practical to depend on autologous blood as a source for fibrinadhesive in trauma cases and other unanticipated surgical emergencies.This invention concerns a convenient and practical method of preparingfibrinogen and fibrin glue which avoids the risk of transmission ofdisease in contrast to prior methods, e.g., the method of Schwarz, etal. This method makes available an abundance of fibrinogen concentratesafe for use within minutes whenever needed in the operating room.

SUMMARY OF THE INVENTION

This invention concerns a method of preparing a cryoprecipitatedsuspension containing fibrinogen and Factor XIII useful as a precursorin the preparation of a fibrin glue. The method involves (a) freezingfresh frozen plasma from a single donor, e.g. a human or other animalsuch as a cow, pig or sheep, which has been screened for bloodtransmitted diseases, e g. one or more of syphilis, hepatitis oracquired immune deficiency syndrome at about -80° C. for at least about6 hours, preferably for at least about 12 hours; (b) raising thetemperature of the frozen plasma, e.g. to about 0° C. to about roomtemperature, preferably to about 4° C., so as to form a supernatant anda cryoprecipitated suspension containing fibrinogen and Factor XIII; and(c) recovering the cryoprecipitated suspension, e.g. by decanting thesupernatant. The suspension may be concentrated, e.g. by centrifugation.

This invention further concerns the cryoprecipitated suspensioncontaining fibrinogen and Factor XIII so prepared, which additionallymay be pre-formed and stored frozen.

The invention also concerns a method of preparing a fibrin glue usefulin surgical procedures which involves (a) preparing a cryoprecipitatedsuspension as described above; (b) applying a defined volume of thesuspension to a desired site; and (c) applying a composition containinga sufficient amount of thrombin from an appropriate source, e.g. human,bovine, ovine or porcine thrombin, to the site so as to cause thefibrinogen in the suspension to be converted to the fibrin glue whichthen solidifies in the form of a gel.

The thrombin-containing composition may also contain a suitable amountof an anti-fibrinolytic substance, such as apoprotinin and may alsocontain CaCl₂.

The suspension and the composition containing thrombin may be applied tothe site in a surgically acceptable vehicle such as a gel, e.g. gelatinor a stretched fabric.

The invention further concerns a method of sealing a surgical woundwhich involves applying to the wound a suitable amount of acryoprecipitated suspension prepared in accordance with this inventionand applying a composition containing thrombin to the site so as tocause the fibrinogen in the suspension to be converted to a fibrin gluewhich then solidifies in the form of a gel. Pressure may be applied tothe fibrin glue until it solidifies. The suspension and composition maybe applied in a surgically acceptable vehicle such as a gel.Alternately, they may be applied in a stretched fabric such as asynthetic vascular patch or graft.

Additionally, this invention involves a fibrin glue kit for use inproviding hemostasis during surgery. The kit contains theabove-mentioned cryoprecipitated suspension and a composition containingthrombin. The thrombin-containing composition may additionally contain asuitable amount of an anti-fibrinolytic substance, e.g. apoprotinin, andmay also contain a suitable amount of an appropriate calcium salt, e.g.CaCl₂. The kit may further contain at least one synthetic vascular graftor patch.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: The use of fibrin adhesive exploits the final stage ofcoagulation cascade. The first component which is prepared from freshfrozen plasma (FFP) by cryoprecipitation contains fibrinogen and FactorXIII (FXIII) and is labeled "PREGLUE". The second component, labeled"GLUE ACTIVATOR" contains thrombin and may additionally containApoprotinin and CaCl₂.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, this invention concerns a method of preparing acryoprecipitated suspension containing fibrinogen and Factor XIII usefulas a precursor in the preparation of a fibrin glue which comprises:

(a) freezing fresh frozen plasma from a single donor which has beenscreened for blood transmitted diseases such as one or more of syphilis,hepatitis or acquired immune deficiency syndrome at about -80° C. for atleast about 6 hours, preferably for at least about 12 hours;

(b) raising the temperature of the frozen plasma to between about 0° C.and room temperature, preferably about 4° C., so as to form asupernatant and a cryoprecipitated suspension containing fibrinogen andFactor XIII; and

(c) recovering the cryoprecipitated suspension.

In a preferred embodiment the cryoprecipitated suspension is prepared in50 cc polypropylene centrifuge tubes (Fisher Scientific, Springfield,N.J.) which have been charged with screened, fresh frozen plasma (FFP)from a single donor. The single donor may be a human or an animal, e.g.a cow, sheep, pig, goat, rabbit, guinea pig, rat or mouse so long as thecryoprecipitated suspension is capable of reacting with thrombin from anappropriate source to produce fibrin glue, as disclosed herein.Preferably the fresh frozen plasma is bovine, ovine or porcine. Thetubes are then placed in a freezer at -80° C. for at least twelve hours,and the fibrinogen-containing suspension prepared for use or storage bythawing over several hours at 4° C. The tubes containing the thawedfibrinogen-containing suspension are then centrifuged, e.g., at about1000-2300×G for fifteen to twenty minutes preferably in a refrigeratedcentrifuge. The supernatant is decanted leaving a yellowish precipitatecontaining fibrinogen. The precipitated and centrifuged pellet ofconcentrated fibrinogen is resuspended in the small amount ofsupernatant remaining after decantation.

Since the final concentration of fibrinogen is partly determined by thevolume of residual supernatant which is used to resuspend the fibrinogenpellet, this maneuver is performed with care so as to remove as muchliquid as possible without disturbing the pellet. Next, the concentratedfibrinogen suspension is aspirated into a syringe using a large borespinal needle. The total yield is approximately 2 cc's of concentratedfibrinogen from each 40 cc's of FFP. Measured fibrinogen in thisconcentrate is 2160 mg % compared to 260 mg % in the virgin FFP (seeTable I). Factor XIII also is present in the final preparation. Allaspects of the procedure are performed in a laminar flow hood understerile conditions and the final product is checked for contamination byroutine culture.

The concentrated fibrinogen-containing cryoprecipitated suspension maybe prepared well in advance of its intended use, i.e., pre-formed, andstored for up to two months at -80° C. before use. Reports indicate itcan be stored for as long as one year (5). After thawing, it reportedlycan be kept for three to four days at 4° C., but we have found itretains biological activity for as long as two weeks. Once at roomtemperature, the concentrate should be used within four hours (5).

This invention further involves a method of preparing a fibrin glueuseful in surgical procedures which comprises: (a) preparing acryoprecipitated suspension as described above; (b) applying a definedvolume of the suspension to a desired site; and (c) applying acomposition containing a sufficient amount of thrombin to the site so asto cause the fibrinogen in the suspension to be converted to the fibringlue which then solidifies in the form of a gel.

                  TABLE I                                                         ______________________________________                                        Fibrinogen, Factor XIII, and Fibrin Split Product Levels                      in Representative Samples of Fresh Frozen Plasma and                          Whole Blood                                                                   Sample    FSP (g/ml)                                                                              Factor XIII                                                                              Fibrinogen (mg %)                              ______________________________________                                        FFP-A.sup.1                                                                             --        present     260                                           FFP-A.sup.2                                                                             0         present    2160                                           Whole Blood.sup.3                                                                       0         present    2160                                           ______________________________________                                         .sup.1 A sample of FFP was thawed only and then tested without                cryoprecipitation treatment.                                                  .sup.2 Cryoprecipitate of sample A revealing almost a 10 fold increase of     concentration.                                                                .sup.3 Unit of single donor whole blood obtained from Presbyterian            Hospital Blood Bank where it had been stored for several days at              1-4° C.                                                           

Suitable thrombin for use in this invention may be derived from a humanor an animal, e.g. a cow, sheep or pig and includes e.g. human, porcineor bovine thrombin such as commercial bovine thrombin, e.g. Thrombinar®(Armour Pharmaceutical Co , Kankakee, Ill.). The thrombin-containingcomposition may also contain a suitable amount of an anti-fibrinolyticsubstance, e.g. apoprotinin, and a suitable amount of an appropriatecalcium salt, e.g. CaCl₂.

In a preferred embodiment the glue is prepared as follows: thecryoprecipitated suspension containing fibrinogen and Factor XIII andcommercial bovine thrombin (Thrombinar® Armour Pharmaceutical Co.,Kanakee, Ill.) are placed into separate one cc syringes. A definedvolume of the fibrinogen-containing cryoprecipitated suspension isapplied to a desired site. The composition containing a sufficientamount of thrombin is then applied to the site so as to cause thefibrinogen in the suspension to be converted to the fibrin glue whichthen solidifies in the form of a gel. With a thrombin mixture of 500units/ml, the fibrinogen gels into a fibrin clot in less than oneminute. The glue works most effectively when applied in a relatively dryfield. For example, application to a vascular suture line severalminutes prior to clamp removal allows time for the glue to congeal. Atransparent gel-like film adheres to the suture line and results inhemostasis after the clamps are released. In a wet field, the suspensionand the composition containing thrombin may be applied to the site in asurgically acceptable vehicle. In a wet field a vehicle comprising a gelsuch as gelatin, e.g. Gelfoam® (Upjohn Co.), or microfibrillar collagenwafers (collagen fleece) e.g. Avitine® (Alcon, Inc., Humacao, PuertoRico) is preferred. Preferably the collagen is bovine, ovine or porcinecollagen. In the former embodiment the gel is saturated with thrombinand then impregnated with the fibrinogen. The Gelfoam® acts as a vehicleto hold the fibrin while clotting occurs. To control active bleeding,the glue should be applied on thrombin soaked Gelfoam® and digitalpressure exerted over the site of bleeding for one minute while the gluesets. Care must be taken to avoid suctioning directly over the area ofglue application because this may result in aspirating not only blood,but also soluble glue before it has polymerized.

To pretreat porous vascular grafts, the technique described by Borst (1)may be employed using a vehicle comprising a stretched fabric. In thisembodiment several milliliters of the fibrinogen concentrate is spreadover the outer surface of the stretched fabric. Following thoroughcoating of the graft, the thrombin activating solution is massaged intothe graft. After the graft is inserted, a few additional drops of glueare applied directly over needle holes before clamp removal. Porousgrafts, so pretreated, offer advantages in handling, suturing andlong-term patency.

As with any technique, successful use of fibrin glue depends on properapplication gained through experience. Our adhesive has stopped bleedingaround vascular anastomoses, particularly needle holes and small lineartears, in a variety of situations involving aortotomies, reversesaphenous vein graft aortic and coronary artery anastomoses, and rightventricular conduit suture lines. By helping to control difficultbleeding, the use of fibrin glue in accordance with this invention candecrease the need for blood transfusions, shorten operating room timeand may even be lifesaving. We have found that as experience is gainedwith the glue the list of potential uses is expanding.

Fibrin glue can be prepared from single donor FFP in sufficient quantityto meet surgical demand because the ease of extraction permits makinglarge amounts of the glue. It can be stored for long periods of time at-80° C. or for shorter periods of time at 4° C. until it is needed. Bythe method of this invention, it is not necessary to anticipate anoperation days in advance in order to have fibrin glue available. It hasbecome our practice to maintain a supply of glue at 4° C. for immediateuse. This stock is checked and updated periodically.

One unit of FFP yields eight to ten cc's of concentrated fibrinogen.This represents enough glue to use in several operations. Approximatelytwo to three cc's of concentrated cryoprecipitated suspension containingthe fibrinogen combined with an equal volume of thrombin is adequate topreseal a right ventricular conduit. As little as one cc of glueeffectively achieves hemostasis of bleeding suture lines or needleholes. In comparison, when fibrinogen is prepared from autologous wholeblood, a smaller volume is extracted (approximately one cc for every onehundred cc's of whole blood) and it is limited to the volume of blooddonated by that individual.

Moreover, the method of this invention of preparing the cryoprecipitatedfibrinogen-containing suspension and the fibrin glue is easy to learn,reproducible, and economical. Most importantly, the use of single donorFFP entails no greater risk of transmission of Hepatitis B, AcquiredImmune Deficiency Syndrome, and other serologically transmitted illnessthan transfusion of a unit of fresh frozen plasma. This decreased riskof blood born infection should circumvent the risk of blood transmitteddiseases which led to the FDA proscription against preparing fibrinconcentrate from pooled donors. Fibrin glue in accordance with thisinvention could become as readily available in this country as thepreparation from pooled blood has been in Europe.

Comparison of fibrinogen prepared from autologous whole blood with thelyophilized, commercial European product (Tissucol®, Immune AG, Vienna,Austria) indicates the tear coefficient of the latter is stronger (5).Unlike Tissucol®, one embodiment of the present invention lacksantifibrinolytic additives and calcium chloride in the activatingsolution. The tenfold concentration of fibrinogen and presence ofassayable Factor XIII indicate the hemostatic potency of ourpreparation. When used as a hemostatic agent or graft sealant, theFFP-derived glue functions so well that its adhesive properties areclear and unequivocal.

The cryoprecipitate of this invention can be included in a fibrin gluekit which also includes a composition containing thrombin. Thethrombin-containing composition may additionally include a suitableamount of an appropriate calcium salt such as CaCl₂ or ananti-fibrinolytic substance such as apoprotinin or both. The kit mayalso include a surgically acceptable vehicle for the fibrin glue, suchas a gel, or at least one synthetic vascular graft or patch or both. Inparticular, highly porous knitted grafts which appear to have long termpatency rates superior to other types of grafts, but cannot presently beused in anti-coagulated patients (because of uncontrollable lifethreatening bleeding) could become widely utilized through thistechnique and may be included in a fibrin glue kit of this invention. Italso may be possible to employ smaller synthetic fabric grafts inperipheral vascular surgery, currently not used because of highocclusion rates.

The methods, materials or kits of this invention may be used for sealinga surgical wound by applying to the wound a suitable amount of acryoprecipitated suspension of this invention and applying a compositioncontaining thrombin to the site so as to cause the fibrinogen in thesuspension to be converted to a fibrin glue which then solidifies in theform of a gel. Pressure may be applied to the fibrin glue until itsolidifies. The suspension and composition may be applied in asurgically acceptable vehicle, e.g. a gel. They may also be applied in astretched fabric such as a synthetic vascular graft or patch. Uses forthe methods, materials or kits of this invention in vascular surgeryinclude providing hemostasis for stitch hole bleeding of distal coronaryartery anastomoses; left ventricular suture lines; aortotomy andcannulation sites; diffuse epimyocardial bleeding seen in reoperations;and oozing from venous bleeding sites, e.g. at atrial, caval, or rightventricular levels. The invention is also useful for stopping bleedingfrom damaged spleens (thereby saving the organ), livers, and otherparynchymatous organs; sealing tracheal and bronchial anastomoses andair leaks or lacerations of the lung; sealing bronchial stumps,bronchial fistulas and esophageal fistulas; for sutureless seamlesshealing ("Zipper" technique), and embolization in vascular radiology ofintracerebral AVM's, liver AVM's, angiodysplasia of colon, esophagealvarices, "pumping" GI bleeders secondary to peptic ulcers, etc. Thisinvention is further useful for providing hemostasis in cornealtransplants, nosebleeds, post tonsillectomies, teeth extractions andother applications.

EXAMPLE 1 Preparation of Cryoprecipitated Suspension (Pre-Glue)

FFP or citrated whole blood is obtained at least about 18 hours beforeintended use of Pre-glue. If whole blood is used as the startingmaterial, it is first centrifuged at 2300×G for 5 min in 50 mlpolypropylene centrifuge tubes (Fisher Scientific, Springfield, N.J.).The plasma is then aspirated carefully, so as not to disturb the buffycoat or RBC layer. The plasma so prepared or FFP is then frozen at -80°C. for at least 12 hours. The frozen plasma is then thawed slowly at 4°C. (or at room temperature, though 4° C. is preferable). After thawingthe tubes are centrifuged again at 2300×G for 20 minutes in arefrigerated centrifuge to separate the fluffy cryoprecipitate. Thetubes are removed from the centrifuge carefully, so as not to resuspendthe pellet. The supernatant is carefully decanted. Tapping the tubesresuspends the cryoprecipitate in the remaining plasma. The pellet,which contains fibrinogen and FXIII, is thick and yellow, and isaspirated into a syringe with a spinal needle for use or transfer tostorage. All equipment used in the above-described example must besterile.

EXAMPLE 2 Preparation of Fibrin Glue

A drop of the cryoprecipitated suspension prepared as in Example 1 isplaced at the desired site. An amount of a composition containingreconstituted thrombin (e.g. Thrombinar®, Armour Pharmaceutical Co.,Kankakee, Ill.) appropriate to the amount of fibrinogen used is thenadded to the desired site. The thrombin causes the fibrinogen to beconverted into the fibrin glue which solidifies in the form of a gel inless than one minute.

EXAMPLE 3 Preparation of Bovine Cryoprecipitated Suspension (BovinePre-Glue)

Example 1 may be repeated using fresh frozen bovine plasma or citratedbovine whole blood instead of human FFP or citrated human whole blood.Bovine pre-glue may be thereby obtained.

EXAMPLE 4 Preparation of Bovine Fibrin Glue

Example 2 may be repeated using bovine pre-glue prepared as in Example 3in place of cryoprecipitated suspension of Example 1.

EXAMPLE 5 Use of Fibrin Glue without Anti-Fibrinolytic Additives

Fibrin glue prepared by the method of Example 2 was used to coverexposed saphenous vein grafts in a patient with a sternal woundinfection which was treated with open drainage. The glue was monitoredand was still in place 48 hours after application. The patient hadreceived multiple wet to dry dressing changes and water pick irrigationsof the wound without dislodgement of the glue. No evidence of grossfibrinolysis, e.g. late bleeding, was observed Since the glue was formedwithout an antifibrinolytic additive such as. apoprotinin, suchadditives do not appear to be essential.

References

1. Borst H. G., Haverich A., Walterbusch G., Maatz W.: Fibrin Adhesive:an important hemostatic adjunct in cardiovascular operations. J. Thorac.Cardiovasc. Surg. 84:548-553 (1982).

2. Bove J. R.: Fibrinogen--Is the benefit worth the risk? Transfusion18:129-136 (1978).

3. Brands W., Mennicken C., Beck M.: Preservation of the ruptured spleenby gluing with highly concentrated human fibrinogen: Experimental andclinical results. World J. Surg. 6:366-368 (1982).

4. Conte J. E., Hadley W. K., Sande M., The University of California,San Francisco, Task Force on the Acquired Immunodeficiency Syndrome:Infection-control guidelines for patients with the acquiredimmunodeficiency syndrome (AIDS). N. Engl. J. Med. 309:740-744 (1983).

5. Gestring G. F., Lerner R.: Autologous fibrinogen for tissue adhesion,hemostasis and embolization. Vascular Surgery 17:294-304 (1983).

6. Glynn M. F. X., William W.: A technique for preclotting vasculargrafts. Ann. Thorac. Surg. 29:182-183 (1980).

7. Guidoin R., Snyder R., Martin L., Botzko K., Marois M., Awad J., KingM., Domurado D., Bedros M., Gosselin C.: Albumin coating of a knittedpolyester arterial protheses: An alternative to preclotting. Ann.Thorac. Surg. 37:457-465 (1984).

8. Haverich A., Oelert H., Maatz W., Borst H. G.: Histopathologicalevaluation of woven and knitted dacron grafts for right ventricularconduits: A comparative experimental study. The Annals of Thoracic Surg37:404-411 (1984).

9. Haverich A., Walterbusch G., and Borst H. G.: The use of fibrin gluefor sealing vascular prostheses of high porosity. Thorac. Cardiovasc.Surgeon 29:252-254 (1981).

10. Kalmar P., Krebber H. J., Pokar H., Tilsner V.: Bioadhesive incardiac and vascular surgery. Thorac. Cardiovasc. Surg. 30:230-231(1982).

11. Kratz J. M.: Preclotting vascular grafts (Correspondence). Ann.Thorac. Surg. 31:97 (1981).

12. Meisner H., Struck E., Schmidt-Habelman P., Sebening F.: Fibrin sealapplication. Clinical experience. Thorac. Cardiovasc. Surg. 30:232-233(1982).

13. Revocation of Fibrinogen Licenses: FDA Drug Bulletin 8:15 (1978).

14. Scheele J., Gentsch H. H., Matheson E.: Splenic repair by fibrintissue adhesive and collagen fleece. Surgery 95:6-13 (1984).

15. Thurer R. L., Hauer J. M., Weintraub R. M.: A comparison ofpreclotting techniques for prosthetic aortic replacement. Circulation66:I-143-I-146 (1982).

16. Walterbusch G., Haverich A., Borst H. G.: Clinical experience withfibrin glue for local bleeding control and sealing of vascularprostheses. Thorac. Cardiovasc. Surg. 30:234-235 (1982).

17. Wolner E.: Fibrin gluing in cardiovascular surgery. ThoracCardiovasc. Surg. 30:236-237 (1982).

18. Yates S. G., Aires M. S., Barros D'SA M. B., Berger K., Fernandez L.G., Wood S. J., Rittenhous E. A., Davis C. C., Mansfield P. B., SauvageL. R.: The preclotting of porous arterial prostheses. Ann. Surg.188:611-622 (1978).

19. "Do-It-Yourself" Fibrin Glue, News Bulletin/American College ofSurgeons, pg. 8, February 1984.

What is claimed is:
 1. A method of preparing a cryoprecipitatedsuspension containing fibrinogen and Factor XIII useful as a precursorin the preparation of a fibrin glue which consists essentially of:(a)freezing fresh frozen plasma from a single donor which has been screenedfor blood transmitted diseases at about -80° C. for at least 6 hours,(b) raising the temperature of the frozen plasma so as to form asupernatant and a cryoprecipitated suspension containing fibrinogen andFactor XIII, and (c) recovering the cryoprecipitated suspension.
 2. Amethod of claim 1, wherein the donor is a human or other animal.
 3. Amethod of claim 2, wherein the donor is a cow, sheep or a pig.
 4. Amethod of claim 1, wherein the blood transmitted diseases comprise oneor more of syphilis, hepatitis or acquired immune deficiency syndrome.5. A method of claim 1, wherein the fresh frozen plasma is frozen instep (a) for at least about 12 hours.
 6. A method of claim 1, whereinthe temperature of the frozen plasma is raised in step (b) to betweenabout 0° C. and about room temperature.
 7. A method of claim 1, whereinrecovering the cryoprecipitated suspension comprises decanting thesupernatant from the cryoprecipitated suspension.
 8. A method of claim 1which further comprises concentrating the cryoprecipitated suspension bycentrifugation.